MIF活性区域相互作用蛋白的筛选

目的: 筛选与巨噬细胞移动抑制因子(MIF)催化巯基蛋白质氧化还原酶(TPOR)活性域相互结合的蛋白。方法: 采用酵母双杂交系统进行筛选。首先构建pBTM116-MIF诱饵质粒,转化酵母菌株L40;将表达MIF催化 TPOR活性域的L40酵母菌株制备成感受态菌,在人骨肉瘤cDNA文库中筛选。通过组氨酸(HIS3报告基因活性和β半乳糖苷酶实验,初步确定与催化TPOR活性域片段相互作用的蛋白,最终通过免疫共沉淀和免疫荧光技术来确认。结果: 分别构建含野生型MIF的pBTM116-MIF和野生型硫氧还蛋白样蛋白2(TXNL2)的pACT2-TXNL2质粒,将其共转染L40酵母菌。HIS3报告基因活性检测和β半乳糖苷酶阳性实验结果提示TXNL2可能是与MIF催化 TPOR活性片段相互作用的蛋白。将重组质粒pcDNA3.1-Myc-TXNL2转染MCF7细胞,免疫共沉淀实验结果表明MIF抗体能够共沉淀Myc-TXNL2蛋白;免疫荧光结果显示Myc-TXNL2与MIF在细胞质中位置分布相同。结论: TXNL2可能是与MIF催化TPOR活性片段相互作用的蛋白。本研究为MIF氧化还原机制的研究提供了新的实验依据。

Screening of proteins interacting with activation domain of macrophage migration-inhibitory factor

AIM: To screen the proteins that interact with the catalytic thiol-protein oxidoreductase (TPOR) domain of macrophage migration-inhibitory factor (MIF). METHODS: Yeast two-hybrid system was used to study the proteins that interacted with MIF. The bait vector pBTM116-MIF was constructed and transfected into L40 yeast strain. L40 competent cells expressing the TPOR domain were prepared and used to screen the proteins interacting with the TPOR domain of MIF in a human osteosarcoma cDNA library. The proteins interacting with TPOR domain were identified by HIS3 reporter gene and β-galactosidase assay. The techniques of co-immunoprecipitation and immunofluorescence were also used to verify the proteins interacting with TPOR domain. RESULTS: Wild-type pBTM116-MIF and pACT2-TXNL2 (thioredoxin-like 2 protein) were constructed and co-transfected into L40 yeast strain. The activation of HIS3 reporter gene and the positive result of β-galactosidase assay indicated that TXNL2 was the candidate protein that interacted with the catalytic TPOR domain of MIF. The vector of pcDNA3.1-Myc-TXNL2 was constructed and transfected into MCF-7 cell line. The result of co-immunoprecipitation assay showed that the catalytic TPOR domain of MIF co-immunoprecipitated with Myc-TXNL2 protein. The result of immunofluorescence assay demonstrated that the catalytic TPOR domain of MIF and TXNL2 co-localized in the cytoplasm of the cells. CONCLUSION: TXNL2 is the protein that interacts with the catalyzed TPOR domain of MIF.